The invention relates to a device for continuous annular chromatography sterilizable in an autoclave, and to a method for using the device.
From WO 99/12625, an annular chromatograph is known which has a stationary eluate collecting ring formed as a slide ring on which a rotating body comprising the chromatographic gel particle bed in the shape of a cylinder jacket and an integrated bottom plate is slidingly and sealingly guided. The purpose being to protect continuous annular chromatography (international abbreviation CAC) performed with this device against contamination by the environment and to enable sterile work. Collecting points in the form of chambers with liquid withdrawal are arranged in the eluate collecting ring. Channels receiving the fractions draining from the particle bed of the CAC column and conveying them to the chambers of the eluate collecting ring are present in the bottom plate.
Particularly for separations of biological or biochemical materials and in applications for the working up of pharmaceutically active substances, the requirements for a chromatographic process free of contamination are stringent. Systems closed toward the outside are required here which are readily sterilized and in addition are readily adapted to changing separation jobs, while it must be remembered that the device that is to be operated free of contamination consists of rotating and stationary parts. In addition, a need exists for provisions being taken during eluate withdrawal that the discharge of the eluate or fractions from the particle bed and the chambers in the collecting ring be as uniform as possible and at the same time the separation as sharp as possible. Moreover, appropriate measures should be taken so that fractions already separated will not later be xe2x80x9cspreadxe2x80x9d to neighboring chambers.
According to WO 99/12625, the desire for improved discrimination in the separation was met by design, by raising the number of channels in the bottom plate and of chambers in the collecting ring to a maximum that was technically feasible and represented an economically meaningful maximum. The resulting problems caused by effects of adhesion and capillarity due to the reduced cross sections of the channels in the bottom plate and particularly of the liquid withdrawal ducts of the collecting ring chambers were solved by increasing the diameter of the channels in the bottom plate radially upward toward the particle bed so as to make them wedge-shaped. The chambers were additionally provided with pressure equalization ports that for sterile operation can be covered up by suitable sterile filter membranes, in order to prevent the development of underpressure and resulting aspiration of nonsterile foreign air into the chambers via the slide ring packing.
The undesired spreading of parts of the fractions into neighboring chambers of the collecting ring was reduced according to WO 99/12625, primarily by selecting a diameter of the channels in the bottom plate that was considerably larger than the thickness of the radial walls between the chambers of the collecting ring. The liquid stream arriving from a channel is then cut off by the radial walls as with a shear blade when the channel slides over such a radial wall, so that the liquid column existing between channel and chamber which is important for a regular liquid drain and which is substantially thicker than the radial wall between two neighboring chambers, is interrupted only in part but never over its entire cross section.
The present invention which primarily represents a development of the CAC device from WO 99/12625 but is not restricted to the features of this document refers to a device for annular chromatography with a stationary column head, a stationary eluate collecting ring, a rotating body between head and ring which is rotatable about a vertical axis and consists of a twin cylinder forming a hollow jacket filled with the chromatographic separation medium, and a mechanical drive. At its bottom end the rotating body is connected with a rotating platform which has vertical through channels and is guided sealingly and slidingly on a stationary eluate collecting ring with chambers. The rotating platform has an extension at its underside that can be coupled with a drive shaft of the drive by a rapidly detachable connection. The stationary column head, the rotating body with the rotating platform and the adjacent eluate collecting ring are combined into a CAC module and can be lifted as a unit from the drive shaft.
The rotatable and stationary parts of the device according to the invention form a unit that is independent of the drive and is held together by suitable means. The rotating body and the stationary parts of the device without the drive unit can be clamped into a kind of cage, for instance between plates, in order to secure a durable union, particularly of the sliding seal between the rotating body and the eluate collecting ring, even during manipulations such as removal from the drive or transport.
It is the purpose of this measure to have a chromatography device which together with the rotating body, the stationary column head and ports for liquid feed lines, the eluate collecting ring, the liquid drains of the chambers in the eluate collecting ring and, where applicable, the eluate collection vessel can be removed as a unit from the drive block, for instance, in order to sterilize it in an autoclave. However, the CAC device according to the invention preferably contains as well all design elements needed for a superheated-steam sterilization in situ, in the manner known particularly from fermentation technology, by passing saturated steam while the device is attached to the drive block. For a sterile process, preferably both the ducts feeding liquid to the column head and the ducts draining the chambers of the eluate collecting ring are provided with sterile connectors. For the feed ducts, for instance, standard sterile connectors such as the ones known from fermentation technology can be used. Additional sealing can also be used, particularly at the seams or contact points between components of the rotating body.
Shafts, V-belts, driving chains, toothed-wheel gearing and friction gear are suitable as a drive. The V-belt pulleys or gear rims can be mounted directly at the periphery of the rotating body; toothed wheels and friction wheels can also act on the inner wall of the rotating body having the shape of a cylinder jacket.
Drive shafts are preferred onto which the rotating body can be placed and to which it can be coupled. It is preferred here to couple the rotating body to the drive shaft, directly via a bottom plate shaped as a rotating platform immediately adjacent to the particle bed. To this end, the rotating platform has a short extension at its underside which is essentially of the same shape as the drive shaft and terminates into a sleeve which can be aligned with and pushed over the drive shaft so as to accept the torque of the drive shaft. Known torsion connectors and securities against twisting of the shafts can be used for coupling. Rigid coupling connectors for aligned shafts or radial serrations can for instance be used. What matters is that the rotating body can be removed from the drive shaft in a reversible and simple fashion without any demanding assembly work, and that the torque is reliably transmitted. Other coupling connectors suitable for connecting the rotating body to the drive shaft are known to one skilled in the art, and include plug-in, snap, slide-lock, catch, and screw connections.
At its coupling end, the drive shaft can be shaped in known fashion, for instance as a cylinder, cone, or polyhedron; the extension of the rotating platform to be attached to it must then be provided with an aligned sleeve having a complementary shape. Conversely, the drive shaft can terminate in a sleeve into which the extension of the rotating platform is precisely fitted with its end having a complementary shape.
The slide ring seal between the rotating platform and the stationary eluate collecting ring must always be compressed as needed so that the rotating body will be guided sealingly on the eluate collecting ring. The rotating body""s own weight can provide the needed pressure. However, it is preferred according to the invention that one or several prestressed compression or tension springs are used. When prestressed tension springs are used, the eluate collecting ring is hung into them; when compression springs are used, they exert pressure from below on the eluate collecting ring. It is advantageous to provide pressure transfer pads with a pressure surface larger than the springs between the springs and the eluate collecting ring in order to distribute the spring force uniformly over the section of the eluate collecting ring.
The slide ring seal, the eluate collecting ring with its embedded chambers receiving the eluates draining from the particle bed, as well as the channels in the bottom plate or rotating platform can be realized in the fashion described initially, and known from WO 99/12625. However, in the present invention it is preferred to solve the problem of pressure fluctuations or underpressure described initally for the CAC device of WO 99/12625 in another way than by using pressure equalization ports in the chambers of the collecting ring.
In fact, it has surprisingly be found that hoses used as eluate withdrawal ducts from the chambers of the eluate collecting ring which where appropriate can be pushed over outlet sockets present at the chambers will solve the underpressure problem even without pressure equalization ports in the chambers, provided the hoses coming from the chambers or outlet sockets are made into siphons, by being for instance bent into a wave shape similar to a recumbent xe2x80x9cSxe2x80x9d. It is unimportant then whether the hoses or eventual outlet sockets are attached to the bottom of the chambers and essentially point vertically down, or whether they are laterally attached to the chambers and preferably point outward and obliquely downward.
In this connection, it is decisive for optimum function of the CAC device according to the invention that between the particle bed and the siphons of the draining hoses a liquid through connection as in communicating vessels arises which despite rotation of the particle bed is preserved throughout the phases of elution and, where applicable, of washing and regeneration. For this reason it is not necessary either to keep the ends or highest portions of the draining hoses at a level below the chambers of the collecting ring, they rather canxe2x80x94and preferably shouldxe2x80x94be lifted to a level higher than the collecting ring and maintained there.
Apart from the invention being adapted for a sterilization in an autoclave, the modular structure of the device offers the advantage of simple interchangeability of the entire chromatography unit. Depending on the type of separation problem being dealt with, rotating bodies fully prepared can without waiting be placed one after the other onto the drive as complete chromatography units (modules) so that during a preparative CAC process, another module can already, for instance, be filled and conditioned in parallel.
It is not only possible to operate one and the same drive block with alternating chromatography modules, one also can adapt the hollow cylinder jackets containing the separation medium and the eluate outlets to the particular chromatographic job. For instance, hollow cylinder jackets or rotating bodies can be varied in their length by using the appropriate spacer rods of different length. The invention is also suited for compound bodies where several particle beds on top of each other or next to each other are used simultaneously for a separation, such as for instance described in WO 99/47913. By analogy, the basic idea and the principle of the present invention can be applied quite generally to all variants of particle beds and CAC devices such as disclosed in the international patent applications WO 98/45699, WO 99/12625, WO 99/29388, WO 99/28740, and WO 99/47913. The term xe2x80x9cparticle bedxe2x80x9d should be understood here, not exclusively as a particulate chromatography gel or resin arranged in the hollow cylinder jacket of the rotating body but also as a monolithic separation medium suitable for liquid chromatography, for instance made of ceramic, plastic (for instance block polymer), fiber material and the like. The term also refers to a combination of two or several chromatographic separation media, where necessary in superimposed layers, preferably with separating layers in between, such as known from the precited publications.